Nanocomplexes of polyanion-modified proteins

ABSTRACT

A nanocomplex, 50 to 1000 nm in size, containing a lipid-like nanoparticle formed of a cationic lipid-based compound and a modified protein formed of a protein and an anionic polymer that includes a plurality of polar groups, the lipid-like nanoparticle and the modified protein being non-covalently bonded to each other. Also disclosed are a method of preparing the above-described nanocomplex and use thereof for treating a medical condition. Further disclosed is a pharmaceutical composition containing a nanocomplex.

This invention was made with government support under grant 1452122awarded by the National Science Foundation. The government has certainrights in the invention.

BACKGROUND

Protein-based therapeutics have received great attention due to theirhigh specificity and low off-target effects. They are widely used fortreating various medical conditions, including cancer, infection,diabetes, inflammation, and degenerative diseases.

Currently, most protein-based drugs treat diseases by targeting cellsurface ligands or extracellular domains. Use of protein drugs to targetintracellular sites remains a significant challenge as the cell membraneis largely impermeable to proteins.

Target-specific delivery systems have been developed for transportingprotein-based therapeutics into cells to target a specific site. SeePlace et al., Molecular Therapy-Nucleic Acids, 1, e15 (2012). Examplesof the delivery systems include polymers and inorganic nanoparticles.See Gonzles-Toro et al., Journal of American Chemical Society, 134,6964-67 (2012). Yet, when delivering protein drugs, these systems do notproduce desired therapeutic effects as proteins are not readily releasedinto the cells effectively. See Brown, Expert Opinion on Drug Delivery,2, 29-42 (2005).

There is a need to develop a new system that efficiently delivers aprotein-based therapeutic to its intracellular target site.

SUMMARY

An aspect of the present invention is a nanocomplex that effectivelytransports proteins into cells to exert therapeutic effects. It contains(i) a lipid-like nanoparticle formed of a cationic lipid-based compoundand (ii) a modified protein formed of a protein and an anionic polymercontaining a plurality of polar groups, each being CO₂H, CO₂ ⁻, SO₃H,SO₃ ⁻, PO₃H, or PO₃ ⁻. The lipid-like nanoparticle binds to the modifiedprotein via non-covalent interaction to form the nanocomplex, which hasa particle size of 50 to 1000 nm.

Typically, the cationic lipid-based compound is formed from anelectrophile and an amine. The electrophile can be an epoxide, anacrylate, or an acrylamide and the amine can be a primary or secondaryamine Examples of the protein include a protein-based cytotoxin, anantibody, a transcription factor, or a genome-editing protein; andexamples of the anionic polymer include hyaluronic acid, heparin, DNA,RNA, polysialic acid, polyglutamic acid, pentosan polysulfate sodium,sulphated polysaccharide, negatively charged serum albumin, negativelycharged milk protein, synthetic sulphated polymer, polymerized anionicsurfactant, or polyphosphate.

Another aspect of this invention is a method of preparing thenanocomplex described above. The method includes the following steps:activating an anionic polymer that contains a plurality of polar groups,the polar groups being CO₂H, CO₂ ⁻, SO₃H, SO₃ ⁻, PO₃H, or PO₃ ⁻;conjugating the activated anionic polymer to a protein via covalentbonding to form a modified protein; obtaining a lipid-like nanoparticleformed of a cationic lipid-based compound; and finally bonding thelipid-like nanoparticle to the modified protein to form a nanocomplexhaving a particle size of 50 to 1000 nm.

A still further aspect of this invention is a pharmaceutical compositioncontaining a nanocomplex thus prepared and a pharmaceutically acceptablecarrier thereof.

Finally, this invention further covers a method of using theabove-described nanocomplex for treating a medical condition in asubject, in which the protein contained in the nanocomplex, upondelivery into cells, is released and exerts a therapeutic effect on themedical condition.

The details of the invention are set forth in the description below.Other features, objects, and advantages of the invention will beapparent from the following drawings and detailed description of severalembodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of (i) synthesis of a nanocomplexcontaining hyaluronic acid (HA)-modified RNase A and a cationiclipid-based compound, i.e., EC16-80; and (ii) targeted delivery ofprotein RNase A into CD44 over-expressing cells.

FIG. 2 is a schematic depiction of concentration-dependent cytotoxicityof RNase A-HA, EC16-80/RNase A, and EC16-80/RNase A-HA with and withoutanti-CD44 antibody pretreatment, against (a) A549 and (b) MCF-7 celllines.

DETAILED DESCRIPTION

Disclosed first in detail herein is a nanocomplex that can be used todeliver a protein-based therapeutic into cells.

To reiterate, the nanocomplex contains a lipid-like nanoparticle and amodified protein formed of a protein and an anionic polymer, thelipid-like nanoparticle bound to the modified protein via non-covalentinteraction to form a nanocomplex having a particle size of 50 to 1000nm. The lipid-like nanoparticle is formed of a cationic lipid-basedcompound. On the other hand, the anionic polymer contains a plurality ofpolar groups.

The cationic lipid-based compound can be prepared by reacting anelectrophile with an amine, the electrophile being an epoxide, anacrylate, or an acrylamide and the amine being a primary or secondaryamine.

The epoxide, acrylate, or acrylamide can contain a C₁-C₂₀ alkyl orC₁-C₂₀ heteroalkyl group. Examples include, but are not limited to,

in which X is O or NH and n is 9-17.

Shown below are examples of the primary or secondary amine:

The term “alkyl” refers to a saturated, linear or branched hydrocarbonmoiety, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, and triacontyl.

The term “heteroalkyl” herein refers to an alkyl moiety containing atleast one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As,Se, and Ge.

Unless specified otherwise, both alkyl and heteroalkyl mentioned hereininclude both substituted and unsubstituted moieties.

The protein contained in the nanocomplex can be a protein-basedcytotoxin (e.g., RNase, saporin, gelonin, ricin chain A, shiga toxinchain A1, or botulinum neurotoxin), an antibody, a transcription factor,or a genome-editing protein (e.g., Cas9 or Cpf1).

The anionic polymer used to modify the protein is typically one of thefollowing: hyaluronic acid, heparin, DNA, RNA, polysialic acid,polyglutamic acid, pentosan polysulfate sodium, sulphatedpolysaccharide, negatively charged serum albumin, negatively chargedmilk protein, synthetic sulphated polymer, polymerized anionicsurfactant, and polyphosphate. It should be pointed out that a proteincan be modified with one or more anionic polymers, of which increasedcharge density on the modified protein facilitates the process ofcomplexing it with a lipid-like nanoparticle via non-covalentinteraction (e.g., electrostatic interaction) to form a nanocomplex.

In one embodiment of the nanocomplex described above, the cationiclipid-based compound is formed from an epoxide substituted with C₁-C₂₀alkyl or C₁-C₂₀ heteroalkyl and a primary or secondary amine, theprotein is a protein-based cytotoxin, an antibody, a transcriptionfactor, or a genome-editing protein, and the anionic polymer ishyaluronic acid, heparin, DNA, RNA, polysialic acid, polyglutamic acid,pentosan polysulfate sodium, sulphated polysaccharide, negativelycharged serum albumin, negatively charged milk protein, syntheticsulphated polymer, polymerized anionic surfactant, or polyphosphate.

An exemplary nanocomplex contains a lipid-like nanoparticle formed of acationic lipid-based compound, and a modified protein formed of aprotein and an anionic polymer, in which the cationic lipid-basedcompound is obtained from

the protein is RNase, and the anionic polymer is hyaluronic acid.

Also within the scope of this invention is a method of preparing thenanocomplex described above. The method includes four steps: (i)activating an anionic polymer that contains a plurality of polar groups,the polar groups being CO₂H, CO₂ ⁻, SO₃H, SO₃ ⁻, PO₃H, or PO₃ ⁻; (ii)conjugating the activated anionic polymer to a protein via covalentbonding to form a modified protein; (iii) obtaining a lipid-likenanoparticle formed of a cationic lipid-based compound; and (iv) bondingthe lipid-like nanoparticle to the modified protein to form ananocomplex.

The nanocomplex thus obtained has a particle size of 50 to 1000 nm(e.g., 50 to 500 nm, 50 to 300 nm, and 50 to 140 nm).

Further covered by this invention is a pharmaceutical compositioncontaining a nanocomplex thus prepared and a pharmaceutically acceptablecarrier thereof. The pharmaceutical carrier is compatible with thenanocomplex and should not be deleterious to a subject to be treated.

Still within the scope of this invention is a method of using thenanocomplex thus prepared for treating a medical condition in a subject,the method including administering to the subject in need thereof aneffective amount of a nanocomplex, in which the protein contained in thenanocomplex, upon delivery into cells, is released and exerts atherapeutic effect on the medical condition.

“An effective amount” herein refers to the amount of the nanocomplexthat is required to confer a therapeutic effect on the treated subject,e.g., inhibition of cancer cells growth. Effective doses will vary, asrecognized by those skilled in the art, depending on the types ofmedical uses (i.e., treatment of cancer), route of administration,excipient usage, and the possibility of co-usage with other medicaltreatment.

The nanocomplex of this invention can be used in treating variousmedical conditions, e.g., cancer. Examples of the cancer include, butare not limited to, breast cancer, prostate cancer, ovarian cancer, orleukemia.

In one embodiment, the nanocomplex is used to treat a medical condition,in which cells associated with the medical condition have high CD44expression.

Shown in FIG. 1 below is an illustration of using a nanocomplex of thisinvention for targeting CD44 over-expressing cells.

First, anionic polymer hyaluronic acid (HA) is activated with N-hydroxylsuccinimide (NHS) to form an activated anionic polymer, i.e., HA-NHS,which is subsequently conjugated to protein RNase A to afford a modifiedprotein, i.e., RNase A-HA. Next, a cationic lipid-based compound(EC16-80; structure shown in FIG. 1 ) is used to form a lipid-likenanoparticle, which is complexed with the modified protein to obtain ananocomplex, i.e., EC16-80/RNase A-HA. Finally, targeted intracellulardelivery is performed to convey the nanocomplex thus obtained into CD44over-expressing cells, e.g., A549 cells, thereby releasing RNase A tocause cell death.

It should be pointed out that the HA modification plays two importantroles: (i) conjugation of HA to protein RNase A “cages” the primaryamine groups of the lysine residues exposed on the protein surface,thereby increasing the negative charge density of the protein andfacilitating its electrostatic complexation with the lipid-likenanoparticle; and (ii) HA specifically binds to CD44 receptor, which isoverexpressed on the cell surface of solid tumors, and, as such, HAconjugation to RNase A offers great potential for targeted cancertherapy.

A protocol of using nanocomplexes for treating cancer is described inWang et al., Angew. Chem., 126, 2937-2942 (2014).

To practice the method of the present invention, a composition havingthe above-described nanocomplex can be administered parenterally,orally, nasally, rectally, topically, or buccally. The term “parenteral”as used herein refers to subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordi-glycerides). Fatty acid, such as oleic acid and its glyceridederivatives, are useful in the preparation of injectables, as arenatural pharmaceutically acceptable oils, such as olive oil or castoroil, especially in their polyoxyethylated versions. These oil solutionsor suspensions can also contain a long chain alcohol diluent ordispersant, carboxymethyl cellulose, or similar dispersing agents. Othercommonly used surfactants such as Tweens or Spans or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art.

A composition containing the nanocomplex can also be administered in theform of suppositories for rectal administration.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific examples are, therefore, tobe construed as merely illustrative, and not limitative of the remainderof the disclosure in any way whatsoever. The publications cited hereinare incorporated by reference in their entirety.

Provided below are materials and methods used for preparing,characterizing, or utilizing the nanocomplexes of this invention, whichare described in EXAMPLES 1-4 also below.

Materials

Unless noted otherwise, all chemicals for lipidoid (i.e., a cationiclipid-like compound) synthesis and protein modification were purchasedfrom Sigma-Aldrich or Alfa-Aesar and used directly. Bovine pancreaticribonuclease A (RNase A) was purchased from Sigma-Aldrich. Hyaluronicacid (Research Grade) was purchased from Life Core Biomedical.1-Ethyl-3-(3-diemthylaminopropyl) carbodiimide (EDC) and N-hydroxylsuccinimide (NHS) were purchased from Sigma-Aldrich. Protein activity ofRNase A and RNase A-HA were measured using RNaseAlert® Kit (IntegratedDNA Technologies, Inc., IA). Lipidoid EC16-80 was synthesized throughthe ring-opening reaction of 1,2-epoxyoctadecane andN,N-dimethyl-1,3-propanediamine according to literature reports. Forexample, see Altinoglu et al., Nanomedicine, 2015, 10, 643-657.Bicinchoninic acid (BCA) protein assay reagents were purchased fromThermo Scientific. Commercially available lipids used forlipidoid/protein nanocomplex formulations (DOPE andC16-PEG2000-ceramide) were purchased from Avanti Polar Lipid, Inc.Monoclonal anti-CD44 antibody was purchased from Sigma-Aldrich (C7923).

Cell Lines and Cell Culture

Human breast adenocarcinoma (MCF-7) cells with low CD44 expression andhuman lung adenocarcinoma epithelial (A549) cells with high CD44expression were purchased from ATCC (Manassas, Va., USA). All cell lineswere cultured in Dulbecco's modified Eagle's medium (DMEM;Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1%penicillin-streptomycin (Life Technologies) under an atmosphere of 5%CO₂/air at 37° C.

BCA Test and SDS-PAGE Analysis

The protein concentration of RNase A-HA was determined using a PierceBCA protein assay kit (Thermo Scientific Cat. No. 23227) according tothe manufacturer's instructions. SDS-PAGE analysis was conducted alsoaccording to the manufacturer's instructions using 4-12% Bis-Tris gel(NuPAGE) and Colloidal Blue Staining Kit (Invitrogen, Cat. No. LC6025).

MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometry was employed to analyze the molecular weights ofprotein before and after the chemical modification, RNase A and RNaseA-HA. Samples were prepared by mixing 1μL, of protein solution (1 mg/mL)with 9 μL of matrix solution (saturated sinapinic acid in 50/50acetonitrile/water with 0.1% trifluoroacetic acid (TFA)).

¹H NMR Analysis

Compositions of HA, RNase A, and RNase A-HA were characterized by ¹H NMRanalysis, using D₂O as the solvent and a Bruker AVIII 500 MHz NMRspectroscopy.

Protein Activity Assay

Protein activities of RNase A and RNase A-HA were measured via theRNaseAlert® Kit according to the manufacturer's instructions. Briefly, 5μL RNaseAlert® Substrate and 10 μL assay buffer (provided by the assaykit) were pre-mixed in a 96-well plate. To the above assay substrate, 85μL of RNase A or RNase A-HA (2 ng/mL) was added. Fluorescence intensityof the protein-containing substrate was monitored within 25 minutes at520 nm (excited at 490 nm).

Lipidoid Nanoparticle Formulation

Lipidoid/protein (EC16-80/RNase A and EC16-80/RNase A-HA) nanocomplexeswere formulated by a reported thin film hydration method. See Wang etal., Angew. Chem., 2014, 126, 2937-2942. Briefly, lipidoid EC16-80 wasmixed with cholesterol and DOPE (Avanti Polar Lipids) at a weight ratioof 16/4/1 (EC16-80/cholesterol/DOPE) and dissolved in chloroform. Thechloroform was evaporated under vacuum condition, and further dried toform a thin film at the bottom of the vial. The thin film was thenhydrated with a mixed solution of ethanol/sodium acetate buffer (200 mM,pH=5.2, v/v=9/1). The solution thus formed was subsequently addeddropwise to an aqueous solution of C16-PEG2000-ceramide (Avanti PolarLipids; EC16-80/C16-PEG2000-ceramide=16/1, w/w). The resulting solutionwas incubated at 37° C. for 30 minutes before dialysis (MWCO 3,500 Da)against PBS to obtain a lipidoid nanoparticle solution, which was thenmixed with RNase A or RNase A-HA in PBS (25 mM, pH=7.4) at apredetermined weight ratio to prepare formulated lipidoid/proteinnanocomplexes, e.g., EC16-80/RNase A.

Synthesis of FITC-Labeled Protein

RNase A-HA and RNase A were labeled with fluorescein isothiocyanate(FITC) for cellular uptake study. Briefly, 2 mg of protein (RNase A-HAor RNase A) was dissolved in 750 μL of 0.1 M NaHCO₃ buffer solution(pH=9.5), and mixed with 250 μL freshly prepared FITC solution (4 mg/mLin DMSO). The reaction mixture was protected from light and stirred atroom temperature for additional 2 hours, followed by ultrafiltrationusing Amicon® Ultra Centrifugal Filters (MWCO 10,000 Da, Millipore,Mass.) to remove impurities.

In Vitro Cytotoxicity Assay

MTT (i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay was employed to evaluate the cytotoxicity of free protein andlipidoid/protein nanocomplexes. Briefly, 100 μL of A549 or MCF-7 cellsuspension was added into 96-well cell culture plates at a density of5,000 cells per well, and incubated for 24 h at 37° C. in 5% CO₂ priorto transfection. The cells were then incubated with free RNase A-HA,blank EC16-80 nanoparticles, formulated EC16-80/RNase A, andEC16-80/RNase A-HA (with different lipidoid/protein weight ratios) atdifferent protein concentrations for 48 hours. Cell viability wasdetermined by the MTT assay. The absorbance was measured at 570 nm usinga microplate reader. All experiments were performed in quadruplicate.

Effect of CD44 Receptor Blocking on Cytotoxicity

A549 cells (high CD44 expression) and MCF-7 cells (low CD44 expression)were incubated with anti-CD44 antibody (2 μg/mL) for 2 hours prior totransfection. The cells were then washed with PBS and placed back intofresh culture medium. The lipidoid/protein nanocomplexes described abovewere added to the cell culture medium After 48-h incubation, cellviability was measured by MTT assay. All experiments were performed inquadruplicate.

Flow Cytometry Analysis

Free FITC-RNase A-HA, formulated EC16-80/FITC-RNase A andEC16-80/FITC-RNase A-HA were incubated with A549 and MCF-7 cells for 2hours, with or without pretreatment of anti-CD44 antibody. Cellularuptake efficacies were then analyzed by flow cytometry. Morespecifically, A549 and MCF-7 cells were initially seeded for 24 hours ata density of 150,000 cells per well in 12-well cell culture plates.Formulated EC16-80/protein nanoparticles were prepared by mixingFITC-labeled RNase A (FITC-RNase A) or RNase A-HA (FITC-RNase A) in DMEMat a weight ratio of 2/1 (lipidoid/protein), followed by an additional15 minutes of incubation at room temperature protected from light. Thecells were washed twice with PBS buffer and replaced with fresh DMEM(700 μL per well). Free proteins or lipidoid/protein nanoparticles werethen added. After 6-h of incubation at 37° C., the cell culture mediumwas removed, and cells were washed with 2 mL of 0.1% heparin in PBS,followed by PBS. Cells were then harvested using 0.25% (w/v) trypsinsolution and dispersed in 0.4 mL of PBS for immediate flow cytometryanalysis using a BD FACS Calibur (BD Science, CA).

In Vitro Fluorescent Cell Imaging

To perform a confocal laser scanning microscopy (CLSM) imaging, A549cells were seeded in a BDFalcon™ 4-well culture slide at a density of20,000 cells per well, 24 hours before the experiment. Then afluorescent dye labeled lipidoid/protein nanocomplex was added into thecells culture medium and incubated for another 6 hours. Cells werewashed twice using PBS buffer, and stained with4′,6-diamidino-2-phenylindole. CLSM images were captured on LeicaTCS-SP5 (Leica Microsystems).

Example 1: Synthesis and Characterization of Hyaluronic Acid (HA)Modified RNase A

HA modified RNase A (RNase A-HA) was prepared and characterizedfollowing the procedure described below.

Hyaluronic acid succinimidyl succinate (HA-NHS) was first obtained byactivating HA with EDC and NHS in a molar ratio of 1/5.73/3.6(HA/EDC/NHS) in de-ionized (DI) water. RNase A-HA was then prepared byreacting RNase A with an excess of HA-NHS as follows. The pH value ofthe reaction mixture was maintained at 4.7 by addition of 0.1 M NaOH/0.1M HCl. The reaction was allowed to proceed for 24 hours at roomtemperature under a continuous stirring condition. After 24-h, the pHwas adjusted to 7.4 with 0.1 M NaOH/0.1 M HCl and RNase A was added intothe reaction solution to have a HA-NHS/RNase A molar ratio of 5:1. Thereaction mixture thus formed was then maintained with continuousstirring at pH 7.5 at room temperature for 24 hours to afford finalproduct RNase A-HA, which was purified by dialysis (MWCO 15,000 Da)using DI water at 4° C. for 3 days, then subjected to analysis with BCA,SDS-PAGE, MALDI-TOF, and ¹H NMR, and enzyme activity assays, asdescribed above.

The final product, RNase A-HA, was characterized by SDS-PAGE, MALDI-TOF,and ¹H NMR. Due to the increase in molecular weight and decrease incharge density, it was observed that RNase A-HA showed a lagging band,as compared to unmodified RNase A. MALDI-TOF mass spectrometry showedthat the molecular weight of RNase A-HA was increased by about 2,000 Da,as compared to native RNase A (13.7 kDa), indicating that one HAmolecule was conjugated to protein RNase A, taking the molecular weightof HA (2247 Da) into account. ¹H NMR analysis confirmed the presence ofHA moieties contained in RNase A-HA, as the characteristic protonsignals of HA were observed in the ¹H NMR spectrum of RNase A-HA, ascompared to unmodified RNase A. Additionally, enzyme activities of RNaseA-HA and RNase A were measured by a commercially available RNase A kit.It was observed that RNase A-HA remained active although the enzymeactivity was slightly decreased by ˜15%, as compared to native RNase A.

Example 2: Preparation and Characterization of Protein-LoadedNanocomplexes

Nanocomplexes containing RNase A or RNase A-HA and a lipidoid (i.e., acationic lipid-based compound) were prepared and characterized followingthe procedure described below.

A lipidoid (“EC16-80”) was prepared according to the method reported inSun et al., Bioconjugate Chem., 2012, 23, 135-140.

Nanocomplexes formed from EC16-80 and a protein (RNase A or RNase A-HA)were obtained by using a thin film hydration method reported in Wang etal., Angew. Chem., 2014, 126, 2937-2942. Briefly, EC16-80, cholesterol,and DOPE were mixed at a weight ratio of 16:2:1 in chloroform, and thechloroform was then evaporated under vacuum to form a thin layer film.The thin layer film thus obtained was re-hydrated withphosphate-buffered saline, followed by addition of RNase A or RNase A-HAat a weight/weight ratio of 8:1 (EC16-80: protein) and incubation for 30minutes at room temperature to afford EC16-80/protein nanocomplexes.

Shown in Table 1 below are hydrodynamic size (D_(h)), zeta-potential,and protein loading efficiency of pure nanoparticle (lipidoid) andlipidoid/protein nanocomplexes. D_(h) and zeta-potential were determinedaccording to literature reports. For example, see Ahn et al., ScientificReports 3, Article number: 1997 (2013).

TABLE 1 Hydrodynamic size, zeta-potential, and protein loadingefficiency of lipidoid and lipidoid/protein nanoparticles.Zeta-potential Protein loading Nanocomplex D_(h) (nm) (mV) efficiency(%) EC16-80 153.0 ± 3.6 24.2 ± 2.8 / EC16-80/RNase A 141.3 ± 3.2  5.4 ±2.4 80.3 EC16-80/RNase A-HA 134.5 ± 2.7 −21.5 ± 3.5   92.7

As shown in Table 1, the averaged a of EC16-80/RNase A-HA nanocomplexwas found to be about 134.5 nm, identical to the size observed in a TEMimage analysis. Unexpectedly, the size of EC16-80/RNase A-HA nanocomplexwas smaller than that of EC16-80/RNase A (about 141.3 nm) and that ofpure nanoparticle EC16-80 (about 153.0 nm).

The differences in hydrodynamic size of the pure nanoparticle andprotein loaded nanocomplexes indicate that binding the cationic lipidoidnanoparticle with protein (RNase A or RNase A-HA) compressed the purenanoparticle. The EC16-80/RNase A-HA nanoparticles had the densest andmost compact nanostructure, possibly resulting from increased chargedensity of the modified protein.

Furthermore, charge density on the protein surface influences thezeta-potential of a lipidoid/protein nanocomplex. As shown in Table 1above, the zeta-potential of pure lipidoid nanoparticle (EC16-80) wasabout 24.2 mV and, by contrast, complexation with RNase A and RNase A-HAdecreased their zeta-potentials to 5.4 mV and −21.5 mV, respectively.

These results suggest that the complexation of a lipidoid nanoparticleto a protein was driven mainly by electrostatic interaction.

Finally, as also shown in Table 1, the EC16-80/RNase A-HA nanocomplexunexpectedly exhibited a high protein loading efficiency of 92.7%, ascompared to 80.3% exhibited by EC16-80/RNase A nanocomplex.

These results demonstrate that the HA modification of RNase A largelydecreased the charge density on the protein, strengthened thecomplexation of the modified protein with cationic lipidoidnanoparticles, and increased the protein loading efficiency.

Example 3: In Vitro Cytotoxicity of Protein-Loaded Nanocomplexes

Studies were performed using a MTT assay to evaluate the in vitrocytotoxicity of free RNase A-HA, pure EC16-80 nanoparticle,EC16-80/RNase A nanocomplex, and EC16-80/RNase A-HA nanocomplex againstA549 cells (high CD44 expression) and MCF-7 cells (low CD44 expression).

To optimize the lipidoid/protein weight ratio for subsequent studies,nanocomplexes having various lipidoid/protein weight ratios (w/w,EC16-80/RNase A-HA=3/1, 2/1, 3/2, 1/1, 3/4, 1/2) were formulated andevaluated using the cytotoxicity assay described above on A549 and MCF-7cells. When fixing the concentration of RNase A-HA at 4 μg/mL andsequentially increasing the concentration of EC16-80 from 1/2 to 3/1(the ratio of EC16-80/RNase A-HA), cytotoxicity against A549 cellsincreased gradually from about 40% to about 70% and cytotoxicity againstMCF-7 cells increased gradually from about 40% to about 85%. Notably,each of lipidoid EC16-80 and protein RNase A-HA alone showed negligiblecytotoxicity against either cell line. Based on these results, theweight ratio of lipidoid/protein was fixed at 2/1 in the followingstudies.

Shown in FIG. 2 below are concentration-dependent cytotoxicity of RNaseA-HA, EC16-80/RNase A, and EC16-80/RNase A-HA against A549 and MCF-7cells with and without anti-CD44 antibody pretreatment.

As shown in FIG. 2 , the cytotoxicity of lipidoid/protein nanocomplexeschanged in a dose-dependent manner Both the free RNase A-HA and theEC16-80/RNase A nanocomplex exhibited relatively low cytotoxicityagainst A549 cells and MCF-7 cells, with cell viabilities greater than70% under various protein concentrations from 0.5 to 8 μg/mL. On theother hand, EC16-80/RNase A-HA nanocomplex showed significant inhibitionof cell proliferation at protein concentrations higher than 2 μg/mLagainst both A549 and MCF-7 cells. More specifically, at proteinconcentrations of 2 μg/mL, 4 μg/mL, and 8 μg/mL, EC16-80/RNase A-HAnanocomplex unexpectedly exhibited cell proliferation inhibitions ofabout 40%, about 60%, and about 80%, respectively, against both A549cells and MCF-7 cells.

These results demonstrate that HA modified RNase A, i.e. RNase A-HA, wasefficiently delivered into cells by a nanocomplex containing an EC16-80lipidoid nanoparticle and inhibited cell proliferation in adose-dependent manner.

As HA moieties contained in the nanocomplex could specifically bind totrans-membrane glycoprotein CD44 receptor present on the cell surface,EC16-80/RNase A-HA nanocomplex would be capable of targeting CD44+cancer cells. As a matter of fact, cytotoxicity of EC16-80/RNase A-HAnanocomplex was unexpectedly found to be significantly decreased againstA549 cells (high CD44 expression), but not MCF-7 cells (low CD44expression), when cells were all pretreated with anti-CD44 antibody.

More specifically, as also shown in FIG. 2 above, the cytotoxicity ofEC16-80/RNase A-HA nanocomplex against A549 cells was significantlydecreased from about 60% (without anti-CD44 antibody pretreatment) toabout 40% (with anti-CD44 antibody pretreatment) at a proteinconcentration of 4 μg/mL and from about 80% (without anti-CD44 antibodypretreatment) to about 65% (with anti-CD44 antibody pretreatment) at aprotein concentration of 8 μg/mL.

By contrast, the cytotoxicity of EC16-80/RNase A-HA nanocomplex againstMCF-7 cells was negligibly decreased from about 60% (without anti-CD44antibody pretreatment) to about 55% (with anti-CD44 antibodypretreatment) at a protein concentration of 4 μg/mL and was not changedat about 80% (with or without anti-CD44 antibody pretreatment) at aprotein concentration of 8 μg/mL.

While anti-CD44 antibody pretreatment had a negligible effect on MCF-7cells having low CD44 expression, it significantly blocked the CD44receptors expressed on the surface of A549 cell having high CD44expression, thereby decreasing the intracellular delivery efficiency ofEC16-80/RNase A-HA nanocomplex and, as a result, decreasing thecytotoxicity of the nanocomplex.

These results suggest that CD44 receptor-mediated internalizationpartially contributed to the intracellular delivery of cytotoxic RNase Aprotein. Thus, use of the lipidoid/protein nanocomplex of this inventionfor targeted cancer therapy can increase efficacy of protein-basedtherapeutics and reduce their systemic side effects.

Example 4: Fluorescent Analysis and Cell Imaging

Studies were conducted by using flow cytometry and confocal laserscanning microscopy (CLSM) as follows to evaluate cell internalizationefficiencies of free RNase A-HA, EC16-80/RNase A nanocomplex, andEC16-80/RNase A-HA nanocomplex in A549 and MCF-7 cells.

FITC-labeled RNase A (FITC-RNase A) and RNase A-HA (FITC-RNase A-HA)were used in the flow cytometry study. It was observed that A549 cells(high CD44 expression) treated with EC16-80/FITC-RNase A-HA nanocomplexhad the highest fluorescent intensity, as compared to free FITC-RNaseA-HA and EC16-80/FITC-RNase A. Similar results were observed when MCF-7cells were used.

These results indicate that (i) a cationic lipidoid nanoparticle wasessential for intracellular protein delivery and (ii) HA modificationenhanced the cell internalization efficacy of a nanocomplex bystrengthening the complexation between the protein and the lipidoidnanoparticle.

Furthermore, when CD44 receptors on the surface of A549 cells wereblocked by pretreatment with anti-CD44 antibody, the total fluorescenceintensity of A549 cells (high CD44 expression) treated withEC16-80/FITC-RNase A-HA was significantly decreased. By contrast, theantibody pretreatment had a negligible effect on fluorescent intensityof MCF-7 cells (low CD44 expression). These results, consistent withthose from the cytotoxicity studies described in EXAMPLE 3, also suggestthat blocking CD44 receptor on A549 cell surface can decreaseintracellular protein delivery efficacy and lead to less inhibited cellproliferation.

Additionally. CLSM images confirmed that EC16-80/FITC-RNase A-HAnanocomplex had the highest intracellular fluorescence intensity, i.e.highest amount of proteins delivered into cells, as compared to freeFITC-RNase A-HA and EC16-80/FITC-RNase A nanocomplex.

In sum, EC16-80/FITC-RNase A-HA nanocomplex of this invention wasunexpectedly found to be a highly efficient protein delivery system.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Further, from the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the claims.

1-22. (canceled)
 23. A nanocomplex, comprising: i) a protein comprisingan anionic polymer, and ii) a lipid-like nanoparticle, comprising acationic lipid-based compound formed from an amine headgroup, whereinthe amine headgroup is selected from the group consisting of

wherein the protein is non-covalently assembled with the lipid-likenanoparticle; and wherein the nanocomplex is characterized by a particlesize of 50 nanometers (nm) to 1000 nm.
 24. The nanocomplex of claim 23,wherein the protein is a protein-based cytotoxin, an antibody, atranscription factor, or a genome-editing protein.
 25. The nanocomplexof claim 23, wherein the protein is a protein-based cytotoxin, and theprotein-based cytotoxin is an RNase, saporin, gelonin, ricin chain A,shiga toxin chain A1, or botulinum neurotoxin.
 26. The nanocomplex ofclaim 24, wherein the protein is a genome-editing protein, and whereinthe genome-editing protein is a Cas9 or Cpf1.
 27. The nanocomplex ofclaim 23, wherein the protein-based cytotoxin is an RNase.
 28. Thenanocomplex of claim 23, wherein the anionic polymer comprises aplurality of polar groups selected from the group consisting of CO2H,CO2-, SO3H, SO3-, PO3H, and PO3-.
 29. The nanocomplex of claim 23,wherein the anionic polymer is selected from the group consisting ofhyaluronic acid, heparin, DNA, RNA, polysialic acid, polyglutamic acid,pentosan polysulfate sodium, sulphated polysaccharide, negativelycharged serum albumin, negatively charged milk protein, syntheticsulphated polymer, polymerized anionic surfactant, and polyphosphate.30. The nanocomplex of claim 23, wherein the anionic polymer ishyaluronic acid or heparin.
 31. The nanocomplex of claim 23, wherein thecationic lipid-based compound is formed from an electrophile and anamine headgroup.
 32. The nanocomplex of claim 23 wherein theelectrophile is an epoxide, an acrylate, or an acrylamide.
 33. Thenanocomplex of claim 23, wherein the electrophile is an epoxide.
 34. Thenanocomplex of claim 33, wherein the electrophile is an epoxidesubstituted with C1-C20 alkyl or C1-C20 heteroalkyl.
 35. The nanocomplexof claim 23, wherein the electrophile is


36. A pharmaceutical composition, comprising a nanocomplex of claim 0;and a pharmaceutically acceptable carrier thereof.
 37. A method oftreating a medical condition, comprising administering to a subject inneed thereof an effective amount of a nanocomplex of claim
 23. 38. Themethod of claim 23, wherein the medical condition comprises a cancer.39. The method of claim 38, wherein the cancer is breast cancer,prostate cancer, ovarian cancer, or leukemia.
 40. The method of claim38, wherein the cancer is associated with a cell comprising high levelsof CD44 expression.